Plasma membrane damage triggered by benzalkonium chloride and cetylpyridinium chloride induces G₀/G₁ cell cycle arrest via Cdc6 reduction in human lung epithelial cells

Abstract

Quaternary ammonium compounds, including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), are widely used as disinfectants. Increased use of inhalable products containing BAC or CPC has raised concerns for lung toxicity. This study sought to elucidate the microstructure of plasma membrane damage caused by BAC and CPC and the subsequent mechanism by which the damage is mediated, as assessed using two human pulmonary epithelial cell lines (A549 and BEAS-2B). Scanning electron microscopic observation showed that exposure to BAC or CPC for 3 hr reduced the length and density of microvilli on the plasma membrane in A549 cells. Analysis of cell cycle distribution following plasma membrane damage revealed that BAC and CPC promote G₀/G₁ cell cycle arrest in both cell lines. The protein levels of Cdc6, an essential regulator of DNA replication during G₁/S transition, are decreased significantly and dose dependently by BAC or CPC exposure. CPC and BAC decreased the Cdc6 levels that had been increased by a PI3K agonist in A549 cells, and levels of phosphorylated AKT were reduced in response to BAC or CPC. Conversely, exposure to equivalent concentrations of pyridinium chloride (lacking a hydrocarbon tail) induce no changes. These results suggest that plasma membrane damage triggered by BAC or CPC causes Cdc6-dependent G₀/G₁ cell cycle arrest in pulmonary cells. These effects are attributable to the long alkyl chains of BAC and CPC. The reduction of Cdc6 following plasma membrane damage may be caused, at least in part, by diminished signaling via the PI3K/AKT pathway.

INTRODUCTION

Quaternary ammonium compounds (QACs), which constitute a group of cationic surfactants, have specific antimicrobial functions (Wessels and Ingmer, 2013). In recent years, QACs have been used increasingly in various applications, not only in the medical arena but also in household products like cosmetics and for personal hygiene (Ferk et al., 2007). In particular, use of sanitizing products including QACs has increased due to the coronavirus (COVID-19) pandemic (Lazofsky et al., 2022; Fuchsman et al., 2022). Benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), two of the most frequently used QACs, often are incorporated in inhalable medication formulations and hygiene products such as mouthwash, nasal sprays, and nebulizer solutions, where these compounds serve as disinfectants and preservatives. Therefore, there exists an increasing risk of accidental or chronic inhalation exposure to QACs in humans.

In South Korea, between 2000 and 2011, a spike was observed in victims suffering from lung injury due to inhalation of polyhexamethylene guanidine (PHMG) aerosols from humidifiers filled with water containing PHMG as a disinfectant (Kim et al., 2016; Park, 2016). PHMG is a cationic guanidine polymer with antimicrobial activity against both gram-negative and gram-positive bacteria (Wessels and Ingmer, 2013). The hexamethylene disrupts the hydrophobic interior of the membrane and decreases membrane integrity (Wessels and Ingmer, 2013). Nanometer-sized PHMG-loaded aerosol particles easily penetrate deeply into the respiratory tract and are deposited in the pulmonary region (Park, 2016), triggering severe pulmonary disease upon inhalation exposure.

QACs are amphoteric surfactants with one positively charged quaternary nitrogen atom attached to one or more long hydrophobic alkyl chains; the chemical structures of QACs resemble PHMG. Because of their positive charges, QACs bind strongly to the phospholipid bilayer of the plasma membrane; the long alkyl chain then causes damage to the plasma membrane via perturbation of the bilayers (Wessels and Ingmer, 2013). Past research on the effects of QACs focused on inhibition of bacterial activity (Gadea et al., 2017). However, QACs have been reported to disrupt cell membranes not only in bacteria but also in human cells (Ferk et al., 2007). Despite the health concerns associated with the inhalation of these disinfectants (Swiercz et al., 2013; George et al., 2017), not many studies have investigated the effects of QACs on pulmonary tissues. Inhalation of aerosolized QACs such as BAC and CPC has been reported to cause adverse effects to the respiratory system in humans (George et al., 2017; Preller et al., 1996). Similarly, inhalation exposure to BAC has been reported to lead to pulmonary toxicity and inflammation in mice and rats (Kwon et al., 2019; Larsen et al., 2012). Elevated protein levels of pulmonary immune mediators, such as interleukin (IL)-11, transforming growth factor (TGF)-β and chemokine (C-X-C motif) ligand (CXCL)-1, and DNA damage in bronchoalveolar lavage fluid (BALF) of rats repeatedly exposed to BAC suggest that repeated exposure to the respiratory system causes pulmonary inflammation and lung damage (Park et al., 2022). It has been reported that repeated exposure of rats to CPC increases proinflammatory cytokines IL-6, IL-1β and tumor necrosis factor (TNF)-α levels in BALF (Kim et al., 2021). BAC-exposed rats showed strong inflammatory and irritant activity in the lung after 6 hr inhalation (Swiercz et al., 2008). Furthermore, BAC-exposed human bronchial epithelial cells for 2 hr induced genotoxicity at concentrations in commercial nasal preparations (Deutschle et al., 2006). In a recent study, chronic exposure to BAC was shown to promote pulmonary epithelial mesenchymal transition in human epithelial cells (Kim et al., 2020). Previously, we reported that BAC and CPC change the membrane surface tension and fluidity, as assessed using a Langmuir-Blodgett (LB) monolayer, an artificial model of a pulmonary surfactant monolayer; we further showed that these compounds induce apoptosis in human alveolar epithelial cells (Kanno et al., 2020). Although severe inhalation toxicity has been noted, few mechanistic studies have examined the effects of these compounds on the pulmonary region.

Plasma membranes can be damaged by various attacks, such as perturbations, physical damage, and environmental exposure to chemicals. Damaged plasma membrane must be rapidly repaired to avoid cell death. The cell cycle often is arrested in damaged cells to permit repair or wound healing. However, the repair mechanism for plasma membrane damage caused by BAC and CPC has not been examined. In budding yeast, it has been reported that damage to the plasma membrane results in inhibition of DNA replication to permit healing; this inhibition is mediated via degradation of the cell division cycle 6 (Cdc6) protein (Kono et al., 2016). Cdc6, an ATP-binding protein, is an essential regulator of DNA replication and maintenance of cell cycle checkpoint in eukaryotic cells (Kono and Ikui, 2017; Borlado and Méndez, 2008; Sun et al., 2002). Therefore, in mammalian cells also, we speculated that Cdc6 is related to the recovery from plasma membrane damage.

In the present study, we report how the microstructure of the plasma membrane, including microvilli, is damaged by exposure to BAC and CPC, and explore the mechanism whereby such membrane damage leads to changes in the cell cycle.

MATERIALS AND METHODS

Chemicals

The following reagents were used in this study: BAC (CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA); pyridinium chloride (PC) (CAS# 628-13-7, as a control for a QAC lacking a hydrocarbon tail) and horseradish peroxidase (HRP)-tagged anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody (Cat# 015-25473) (FUJIFILM-WAKO, Osaka, Japan); Halt^TM phosphatase inhibitor cocktail and bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA); BD Cycletest^TM Plus DNA Reagent Kit (BD Bioscience, San Jose, CA, USA); LY294002 (Abcam, Cambridge, UK); 740Y-P (Selleckchem, Houston, TX, USA); HRP-tagged anti-mouse immunoglobulin G (IgG) antibody (Cat# PM009-7; MBL, Nagoya, Japan); RIPA buffer containing protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, and HRP-tagged anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-CDC6 antibody (Cat# C42F7), anti-phospho-AKT (Ser473) antibody (Cat# 9271), and anti-AKT antibody (Cat# 2920) (Cell Signaling Technology, Danvers, MA, USA); One-step RT-PCR (reverse transcription-polymerase chain reaction) Master Mix, PVDF (polyvinylidene fluoride) Blocking Reagent, and Can Get Signal^TM Immunostain (Toyobo, Osaka, Japan); ECL prime western detection reagent (GE Healthcare, Buckinghamshire, UK); and NucleoSpin RNA isolation kit (Macherey-Nagel, Düren, Germany). Unless otherwise specified, all other chemicals including CPC (CAS# 6004-24-6) were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The molecular structures of BAC, CPC, and PC are shown in Fig. 1. The BAC used in this study is a mixture of n-alkyldimethylbenzyl ammonium chlorides with various carbon-chain lengths (C₈–C₁₈). Therefore, the concentration of BAC is given in units of μg/mL.

Fig. 1

Structures of the evaluated compounds and polyhexamethylene guanidine (PHMG).

Cell culture

A human lung epithelial cell line, A549, and a human bronchial epithelial cell line, BEAS-2B (BEAS hereafter), were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured at 37°C in a 5% CO₂ atmosphere in Dulbecco’s modified minimum essential medium (DMEM; Life Technologies, Carlsbad, CA, USA). For use as culture medium, DMEM was supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

The concentrations used in this study, BAC (0–10 µg/mL), CPC (0–10 µM), sodium dodecyl sulfate (SDS; 0–150 µM), Triton X-100 (Triton; 0–25 µg/mL), and cytochalasin D (0–20 µM) for A549 cells, and BAC (0–2.5 µg/mL), CPC (0–2.5 µM), SDS (100 µM), and Triton (25 µg/mL) for BEAS cells, were determined based on cytotoxicity assays in our previous study (Kanno et al., 2020) and in this study (Supplemental Fig. 1), and preliminary experiments. These concentrations show no effect on cell viability within the exposure period of each experiment. Control cells were exposed to the vehicle of each reagent and are noted as control or vehicle-treated cells.

Scanning electron microscopy (SEM)

A549 and BEAS cells were plated at 5 × 10⁴ cells/well and pre-cultured overnight on a cover slip placed on the bottom of a well in a 12-well culture plate. A549 and BEAS cells were exposed to BAC, CPC, or PC for 3 hr. A549 and BEAS cells also were exposed to SDS or Triton for 3 hr or to cytochalasin D for 3 and 6 hr to compare the effects of QACs to those of other compounds. After washing with phosphate-buffered saline (PBS), the cells were pre-fixed in 0.1 M phosphate-buffered 2.5% glutaraldehyde solution containing 0.2 M sucrose and 0.1% tannic acid, followed by post-fixation with 1% osmium tetroxide; these steps were conducted at 25°C for 2 hr. After sequential dehydration using a series of solutions of increasing ethanol concentration, the samples were dried using a critical point dryer and observed by SEM (S-4800, Hitachi, Tokyo, Japan).

Cell cycle analysis

A549 and BEAS cells were plated at 5 × 10⁴ cells/well and pre-cultured overnight in 12-well culture plates. The cells were exposed to BAC, CPC, or PC (only A549 cells) for 14 hr. After exposure, the cells were trypsinized and collected. The cells then were stained with propidium iodide (PI) using the BD Cycletest Plus DNA Reagent Kit according to the manufacturer’s instructions. The cell cycle distribution was analyzed by flow cytometry (FACSCanto II, Beckton Dickinson, Franklin Lakes, NJ, USA).

Western blot analysis

For the detection of Cdc6, A549 and BEAS cells were exposed to BAC or CPC for 9 (only A549), or 14 hr. For the detection of phosphorylation of AKT, A549 and BEAS cells were exposed to BAC and CPC for 8 hr. For inhibitor experiments, A549 cells were pre-cultured with 15 μM 740Y-P or 20 μM LY294002 for 1 hr, and the cells then were exposed to BAC or CPC for 14 hr in the continued presence of the respective pre-culture reagent. After washing twice with PBS, cells were lysed with RIPA lysis buffer containing a protease inhibitor, PMSF, sodium orthovanadate, and Halt^TM phosphatase inhibitor cocktail. The lysate was centrifuged at 10,000 g for 5 min at 4°C. Proteins in the supernatant were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions, and electroblotted onto a PVDF membrane. The membrane was blocked with PVDF Blocking Reagent and probed with anti-Cdc6 or anti-phospho-AKT followed by HRP-tagged anti-rabbit IgG antibody or anti-mouse IgG antibody. The immunoreactions on the membrane were visualized using ECL prime western detection reagent. After stripping the phospho-AKT antibody, the membrane was reprobed with anti-AKT. As a final step for all western blotting, each membrane was again stripped and reprobed with HRP-tagged anti-GAPDH antibody. The protein levels were quantified using a Luminescent Image Analyzer (Amersham, GE Healthcare, Boston, MA, USA). Values were normalized to those of GAPDH, a housekeeping protein, in the respective samples.

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR)

A549 cells were exposed to BAC or CPC for 6 hr. Total RNA was extracted using a NucleoSpin RNA isolation kit. RT-PCR was performed with the One-step RT-PCR Master Mix according to the manufacturer’s instructions. cDNA reverse transcription and PCR amplification were performed using a real-time PCR system (QuantStudio 12K Flex, Thermo Fisher Scientific). The following primers were used: CDC6, forward 5’-ACCTATGCAACACTCCCCAT-3’ and reverse 5’-TGGCTAGTTCTCTTTTGCTAGGA-3’; and GAPDH, forward 5’-CGAGATCCCTCCAAAATCAA-3’ and reverse 5’- TTCACACCCATGACGAAC-3’. Data were analyzed using Sequence Detection System Real-Time PCR data analysis software (Version 2.4; Thermo Fisher Scientific). Relative expression of CDC6 was normalized to that of GAPDH using the ΔΔCt method.

Statistical analysis

Data are presented as means ± SEM. Statistical analyses were performed by ANOVA followed by Bonferroni’s post hoc analysis. The statistical significance level was set at p < 0.05.

RESULTS

Morphological changes in BAC-, CPC-, PC-, SDS- or Triton-exposed A549 cells

Scanning electron microscopic observations indicated that untreated A549 cells were rich in membrane ruffling and microvilli on the cell surface (Fig. 2). Interestingly, dose-dependent diminishment of these surface microvilli, and smoothing of the cell surface, were observed early in response to BAC and CPC exposure. In contrast, no obvious changes were observed in PC-exposed cells. These results indicated that BAC and CPC at concentrations of 2.5 μg/mL and 2.5 μΜ, respectively, or higher caused microvillar disruption in A549 cells. Given that distinct changes in microvilli were seen in A549 cells following exposure to BAC or CPC, we speculated that these changes in microvilli might be involved in membrane damage. Therefore, we further examined whether morphological changes in microvilli were also observed in A549 cells exposed to SDS or Triton, which are typical inducers of plasma membrane damage. Notably, SDS- or Triton-exposed A549 cells also exhibited diminished microvilli on the cell surface (Supplemental Fig. 2A), similar to that seen in BAC- and CPC-exposed A549 cells. Furthermore, the loss of microvilli and small protrusions was observed by treatment with cytochalasin D, a potent inhibitor of actin polymerization and reorganization, in A549 cells (Supplemental Fig. 2B). Interestingly, multiple traces of microvilli (black arrowheads) were visible on the cell surface of cytochalasin D-exposed cells in magnified images.

Fig. 2

Scanning electron micrographs of BAC-, CPC-, or PC-exposed A549 cells. A549 cells were plated and pre-cultured overnight on a cover slip placed in a 12-well culture plate. A549 cells were exposed to BAC (2.5, 5 μg/mL), CPC (2.5, 5 μM), or PC (2.5 μM) for 3 hr. Scale bars, 5 μm.

Effects of BAC, CPC, or PC on the cell cycle in A549 cells

The disappearance of microvilli has been reported to occur at an early stage following plasma membrane damage (Zheng et al., 2017). The cell cycle often is arrested in damaged cells to permit repair or wound healing. Thus, to investigate whether cell cycle arrest occurs even with very early stage of plasma membrane damage caused by BAC or CPC, we analyzed the cell cycle distribution of BAC- or CPC-exposed A549 and BEAS cells using flow cytometry. Flow cytometry histograms and the percentage of distribution are shown in Supplemental Fig. 3 and Fig. 3, respectively. As shown in Fig. 3-a, the percentage of A549 cells in G₀/G₁-phase was increased significantly following exposure to BAC or CPC, resulting in decreased percentages in S- and G₂-phase; these effects were dose dependent. On the other hand, PC did not have a significant effect on the cell cycle in A549 cells (Fig. 3-b).

Fig. 3

Distribution of the cell cycle in BAC-, CPC-, or PC-exposed A549 cells. A549 cells were exposed to BAC (0–5 μg/mL), CPC (0–5 μM) (a), or PC (0–5 μM) (b) for 14 hr. The cell cycle distributions were analyzed by flow cytometry following propidium iodide staining as described in the Methods. Data are presented as means ± SEM (n = 3). *, statistically significant difference vs. control group (p < 0.05).

Changes in Cdc6 protein and mRNA levels in BAC-, CPC-, PC-, SDS-, or Triton-exposed A549 cells

Next, we investigated the mechanism by which the cell cycle arrest was caused by plasma membrane damage. Cell cycle progression at G₁/S transition is tightly regulated, primarily by cyclin-dependent kinases. Among cell-cycle regulatory proteins, we focused on Cdc6, a key cell cycle-related protein responsible for cell cycle arrest. Notably, Cdc6 is an essential regulator of the G₁ checkpoint for DNA replication (Borlado and Méndez, 2008). Western blotting and quantitative analyses revealed that the levels of Cdc6 protein in A549 cells were significantly decreased in a dose-dependent fashion compared to control cultures following a 14-hr exposure to BAC or CPC (Figs. 4A-a, b). The Cdc6 levels in A549 cells were also decreased at 9-hr exposure (Supplemental Fig. 4). In contrast, Cdc6 protein levels were not significantly changed in PC-exposed A549 cells compared to control cultures (Fig. 4A-c). Quantitative analysis of transcript levels revealed that CDC6 mRNA was decreased significantly in BAC- and CPC-exposed A549 cells compared to control cultures (Fig. 4B). Exposure to 100 μM SDS, 25 μg/mL Triton, or cytochalasin D, which decreased the length and density of microvilli, resulted in decreased Cdc6 protein levels in A549 cells (Supplemental Fig. 5).

Fig. 4

Cdc6 protein (A) and mRNA expression (B) levels in BAC- or CPC-exposed A549 cells. A549 cells were exposed to BAC (a) or CPC (b) (0–10 μg/mL or 0–10 μM), or PC (c) (0–5 μM) for 14 hr. Cultured cells were processed for immunoblotting as described in the Methods section. Cdc6 protein levels were normalized to those of GAPDH and are presented as a ratio to the control group. For mRNA expression, A549 cells were exposed to BAC or CPC for 6 hr. The expressions of transcripts encoding Cdc6 and GAPDH were analyzed by RT-PCR. Ct values for CDC6 mRNA were normalized to those of GAPDH in the respective samples. Data are presented as means ± SEM (n = 3). *, significantly different from the control group (p < 0.05).

Changes in morphology, cell cycle and Cdc6 protein level in BEAS cells

Furthermore, we examined the responses to BAC or CPC exposure using BEAS cells. In BEAS cells, the microvilli were not observed on the surface (Fig. 5A). There were many lamellipodia, which are projections of the membrane at the leading edge of cells migrating on a flat substratum. Many small dimples (mean diameter 2.1 μm, range 1.5–3.1 μm (n = 10); Fig. 5A, white arrowheads) were observed on the lamellipodia following exposure to 2.5 μg/mL BAC. On the other hand, no dimples were seen on the surfaces of BEAS cells exposed to CPC or PC at concentrations as high as 1.25 and 2.5 μΜ. Intriguingly, Triton (25 μg/mL) treatment was associated with the appearance on the plasma membrane of numerous small granules with diameters of approximately 500 nm, while SDS (100 μΜ) showed no obvious changes compared with the control (Supplemental Fig. 2C).

Fig. 5

Changes in morphology (A), cell cycle (B) and Cdc6 protein level (C) in BEAS cells. (A) BEAS cells were exposed to BAC (1.25, 2.5 μg/mL), CPC (1.25, 2.5 μM), or PC (2.5 μM) for 3 hr. See also the legend in Fig. 2. White arrows and arrowheads indicate small granules and dimples on the lamellipodia, respectively. (B) BEAS cells were exposed to BAC (0–2.5 μg/mL) or CPC (0–2.5 μM) for 14 hr for the cell cycle distributions. See also the legend in Fig. 3. (C) BEAS cells were exposed to BAC (0–2.5 μg/mL) (a) or CPC (0–2.5 μM) (b) for 14 hr. Cultured cells were processed for immunoblotting as described in the Methods section and the legend in Fig. 4. Data are presented as means ± SEM (n = 3). *, significantly different from the control group (p < 0.05).

The exposure to BAC or CPC increases in the proportion of cells in G₀/G₁-phase were also observed in BEAS cells as well as A549 cells (Fig. 5B). Moreover, a significant dose-dependent decrease in Cdc6 levels was also detected in BEAS cells (Figs. 5C-a, b).

Effect of a phosphoinositide 3-kinase (PI3K) inhibitor and agonist on Cdc6 protein levels

To the best of our knowledge, signal pathways in response to plasma membrane damage in higher eukaryotes have been little investigated. However, PI3K is associated with lipid signaling after plasma membrane injury (Horn and Jaiswal, 2019). LY294002, a PI3K inhibitor, has been reported to downregulate Cdc6 protein expression in K562 chronic myeloid leukemia cells (Zhang et al., 2017). To determine whether the reduction of Cdc6 is mediated by PI3K, the effects of LY294002 and 740Y-P, a PI3K agonist, on Cdc6 protein levels were examined in BAC- or CPC-exposed cells. Cdc6 protein levels were significantly decreased in cultures exposed to 5 μg/mL BAC or 5 μM CPC compared to vehicle-treated A549 cells (Fig. 6), consistent with the results shown in Fig. 4A. LY294002 alone also decreased Cdc6 levels (compared to vehicle-treated A549 cells) in a dose-dependent manner (data not shown). Cdc6 levels were decreased further (becoming undetectable) by exposure to a combination of LY294002 and 5 μg/mL BAC or 5 μM CPC; these additional decreases were significant compared to those seen with either compound alone. On the other hand, 740Y-P exposure resulted in significantly increased Cdc6 protein levels compared to vehicle-treated A549 cells. However, the increases in Cdc6 protein levels following exposure to 740Y-P were significantly attenuated when 740Y-P was tested in combination with 5 μg/mL BAC or 5 μM CPC.

Fig. 6

Effects of a PI3K inhibitor and agonist on CDC6 protein levels in BAC- or CPC-treated A549 cells. A549 cells were pre-cultured with 20 μM Y294002, a PI3K inhibitor, or 15 μM 740Y-P, a PI3K agonist, for 1 hr and then were exposed to BAC (5 μg/mL) or CPC (5 μM) in the continuing presence or absence of Y294002 or 740Y-P for 14 hr. Cultured cells were then processed for immunoblotting as described in the Methods section. Cbc6 protein levels were normalized to those of GAPDH and are presented as a ratio to the control. Data are presented as means ± SEM (n = 3). *, significantly different from the control group (p < 0.05); #, significantly different from the LY294002-treated group (p < 0.05); §, significantly different from the 740Y-P-treated group (p < 0.05).

Effects of BAC and CPC on AKT phosphorylation

Decreased PI3K activity is known to result in decreased phosphorylation of AKT at Ser⁴⁷³ (Cross et al., 1995). We therefore examined whether AKT mediates the signal communicating plasma membrane damage triggered by BAC or CPC to the cell cycle. LY294002 exposure resulted in a decrease (compared to 0 hr as a control) in the amount of phosphorylated AKT at 0.5 hr in A549 cells (data not shown); the level of this phosphoprotein returned to that seen in control cells. Next, we examined BAC and CPC to assess the signaling pathway in A549 and BEAS cells. As shown in Fig. 7, exposure to BAC or CPC resulted in a significant decrease in the proportion of phosphoprotein for AKT in both cell lines, although not significantly in BAC-exposed A549 cells. BAC- or CPC-exposure for 14 hr in BEAS cells also decrease phosphoprotein for AKT (data not shown). On the other hand, the protein levels of total AKT and GAPDH did not appear to be affected by BAC and CPC exposure.

Fig. 7

Changes in AKT phosphorylation in BAC- or CPC-exposed A549 and BEAS cells. A549 or BEAS cells were exposed to BAC (5 or 2.5 μg/mL) and CPC (5 or 2.5 μM) for 8 hr, respectively. Cultured cells were processed for immunoblotting as described in the Methods section. Each membrane was probed with anti-phospho-AKT antibody, then stripped and reprobed using anti-AKT antibody.

DISCUSSION

QACs such as BAC and CPC are amphiphilic compounds containing one positively charged quaternary nitrogen atom with one or more long hydrophobic alkyl chains. We previously reported that the cytotoxicity of BAC and CPC in A549 cells is attributable to the interaction between plasma membrane phospholipids and the long alkyl chains of BAC and CPC (Kanno et al., 2020). These alkyl chains have been reported to interact with the plasma membrane via intense interfacial activity (Uematsu et al., 2010). These findings indicate that the plasma membrane is the primary site of damage triggered by BAC and CPC exposure. In this study, SEM analyses revealed that untreated A549 cells had membrane ruffling and numerous microvilli on the cell surface (Fig. 2), as reported previously (Calcabrini et al., 2004). Intriguingly, exposure of these cells to BAC or CPC for 3 hr resulted in dramatic, dose-dependent decreases in the length and number of microvilli. Microvilli are finger-like protrusions composed of linear F-actin core filaments (Figard and Sokac, 2014), and are found primarily on the apical membrane surface of various epithelial cells, including lung cells and intestinal cells (Hirokawa et al., 1982). Microvilli, which express various transporter proteins and ion channels, have an important role in small-molecule and ion movement across the cell membrane, and cellular adhesion (Bennett et al., 2014). The reduction of microvilli was observed in A549 cells exposed to multi-walled carbon nanotubes (Cavallo et al., 2012), and ozone (Alink et al., 1980). In A549 cells exposed to particulate matter 2.5 (PM2.5) water soluble compound, morphology changes, such as microvilli reduction, partial disappearance of lamellipodia and a strengthened cytoskeleton, have been reported to be due to early cytotoxic effects (Tang et al., 2021). Furthermore, the reduced microvilli decreased cell adhesion (Tang et al., 2021). BAC has been reported to cause a loss of microvilli on the corneal surfaces of rabbits (Tonjum, 1975). Otherwise, few studies (to our knowledge) have tested the effects of BAC or CPC on microvilli. A loss of microvilli also was observed in SDS- or Triton-exposed A549 cells (Supplemental Fig. 2A). SDS and Triton, prototypical ionic and nonionic detergents (respectively), have been reported to cause membrane injury (Malik et al., 1983; Boesewetter et al., 2006). Indeed, the disappearance of microvilli has been reported to occur at an early stage in the process of plasma membrane damage (Zheng et al., 2017). Taken together, the results of the present study suggest that the decreases in length and density of microvilli caused by BAC and CPC is an early stage of membrane damage.

Comparison of the plasma membrane morphologies following exposure to QACs and detergents suggested that the plasma membrane surfaces of BAC- and CPC-exposed A549 cells are smoother than those of the SDS- and Triton-exposed cells, with the QACs resulting in a clean loss of microvilli (Fig. 2, Supplemental Fig. 2A). Conversely, the surfaces of cells exposed to Triton were littered with small protrusions, presumably representing the residua of microvilli. A previous report indicated that the activation of Rho-GTPases such as Rac1 and Cdc42 induced the loss of microvilli; however, the surface morphologies of these cells, including the microvillar residua, differed depending on the specific Rho-GTPase that was being activated (Nijhara et al., 2004). These findings suggest that the formation and/or maintenance of microvilli may be impaired by different mechanisms in cells exposed to QACs and Triton. On the other hand, in BEAS cells, many dimples on the lamellipodia were observed in BAC-treated cells. Lamellipodia, one of cell morphological features, are thin sheet-like leading edge protrusions composed of a highly branched actin filament. The most important functions of lamellipodia are cell polarity and cell migration. BEAS cells lacking lamellipodia partially may cause reduced cell polarity and cell migration. In Triton-exposed BEAS cells, numerous small granules appeared on the plasma membrane (Supplemental Fig. 2C). Although these small granules also were observed in untreated cells (Fig. 5A, control; white arrows), these granules were more numerous and distinct on the surfaces of Triton-exposed BEAS cells. Further studies will be needed to characterize these granules; it is conceivable that such structures form on the plasma membrane as a result of the interaction of Triton with the phospholipid bilayer.

We next investigated whether plasma membrane damage caused by BAC or CPC affects the cell cycle in A549 and BEAS cells. The cell cycle often is arrested in damaged cells, such as DNA damage, apoptosis, and endoplasmic reticulum (ER) stress, facilitating (for instance) tissue repair and/or wound healing. Interestingly, the density of microvilli on HeLa cells has been reported to correlate with the cell cycle (Sommi et al., 2021). Cell cycle analyses revealed that BAC and CPC had arrested cell cycles at the G₀/G₁ phase in both A549 and BEAS cells (Figs. 3, 5B). Therefore, we examined the target leading to cell cycle arrest in response to plasma membrane damage. DNA replication during the G₁ phase of the cell cycle is initiated by the association of Cdc6 with the origin recognition complex (ORC) bound to the replication origin, resulting in the recruitment of Cdc10-dependent transcript 1 protein (Cdt1) and mini-chromosome maintenance complex (MCM) proteins 2-7 to form the pre-replicative complex (pre-RC) assembly (Fragkos et al., 2015). As a component of the pre-RC, Cdc6 is an essential regulator of the G₁ phase cell cycle licensing checkpoint (Borlado and Méndez, 2008). Based on these studies, we hypothesized that Cdc6 participates in cell cycle arrest. Indeed, exposure to BAC, CPC, SDS, Triton or cytochalasin D resulted in decreases in the levels of Cdc6 protein in A549 cells (Fig. 4A, Supplemental Fig. 5). Exposure of BEAS cells to BAC or CPC also resulted in the decreases of Cdc6 (Fig. 5C). These results suggested that plasma membrane damage, whether caused by detergents or QACs, results in arrest at the G₀/G₁ checkpoint via the reduction of Cdc6.

We further examined the effects on signal transduction following plasma membrane damage. Enzymes, such as phosphatidylinositol kinases (PI5K and PI3K), sphingomyelinase and phospholipase, are known to generate lipid signaling after plasma membrane injury (Horn and Jaiswal, 2019). Especially, PI3K/AKT signaling is an important mediator of various cell functions, including cell proliferation (Rodon et al., 2013). PI3K regulates the nuclear cell cycle and activates late G₁ phase entry into S phase (Kumar and Carrera, 2007). Therefore, we speculated that the decrease in Cdc6 protein levels seen following exposure to BAC and CPC is mediated via the PI3K/AKT pathway. Indeed, the depletion of Cdc6 protein following exposure to BAC or CPC was counteracted by the PI3K agonist 740Y-P and further attenuated by the PI3K inhibitor LY294002 (Fig. 6), suggesting that BAC and CPC depletion of Cdc6 protein levels is mediated by decreased PI3K signaling. Furthermore, BAC and CPC exposure resulted in decreased proportions of phosphorylated AKT in both cell lines (Fig. 7). We postulate that decreased PI3K activity and the resulting decreases in the phosphorylation of AKT may be responsible, in part, for the cell cycle arrest observed following exposure of cells to BAC or CPC.

PC contains a positive nitrogen but not the long alkyl moiety; therefore, PC can bind plasma surface, but does not damage plasma membrane via perturbation of the bilayers. The evidence that PC has no cytotoxic effect on A549 and BEAS cells (Supplementary Fig. 1) (Kanno et al., 2020) and no changes in plasma morphology (Figs. 2 and 5A) support that PC does not induce plasma membrane damage. PC did not induce a change in the cell cycle (Fig. 3-b) or in Cdc6 protein levels (Fig. 4A-c), possibly because the PC-exposed cells did not require the repair of plasma membrane damage. These results suggest that cells induce cell cycle arrest to repair the plasma membrane damage by long alkyl chain of BAC and CPC.

CPC is a component of commercial mouthwashes and sprays, where the compound typically is present at concentrations of 0.05% (1.5 mM). BAC is a component of various disinfectant sprays, where this compound is present at concentrations of 0.01–0.025% (0.1–0.25 mg/mL). The concentrations of BAC (0–10 μg/mL) and CPC (0–10 μM) used for the experiments described here therefore were lower than those in medical or commercial products.

The mechanisms of BAC toxicity have been reported in several studies of in vitro experiments. In A549 cells cultured with BAC for 24 hr, oxidative stress levels and IL-8 were upregulated (Kwon et al., 2019; Jeon et al., 2019). In BEAS cells, treatment of BAC increased NO production and lipid peroxidation, but not reactive oxidative species (ROS) (Park et al., 2022). Our previous study indicates that the N-acetylcysteine (NAC) suppressed cytotoxicity by BAC, suggesting that ROS are involved in the cytotoxicity of BAC (Kanno et al., 2020). On the other hand, it has been reported that ROS depletion cannot prevent microvilli degradation in human embryonic stem cells (Zheng et al., 2017). Based on the above, it is likely that microvilli degradation occurs in the early stages of cytotoxicity, even in the absence of excessive ROS production, followed by toxic effects such as apoptosis and inflammatory reactions associated with oxidative stress. Further studies are needed to clarify the toxic mechanisms of BAC and CPC.

In conclusion, this work demonstrated that plasma membrane damage caused by BAC and CPC leads to G₀/G₁ cell cycle arrest in human pulmonary epithelial cells. The reduction of Cdc6 is thought to be related to changes in PI3K signaling and the partial inhibition of AKT phosphorylation. Furthermore, no changes in microvilli morphology and Cdc6 protein levels were observed following exposure to PC, a QAC-like compound lacking the alkyl chain, suggesting that the decrease in Cdc6 is attributable to membrane damage caused by the interaction of the alkyl chains of BAC and CPC with phospholipids. These results suggest that the deposition of BAC and CPC aerosols on pulmonary surfaces may trigger plasma membrane damage, resulting in cell cycle arrest in pulmonary epithelial cells. These observations raise concerns about potential human exposure to QACs used in medical and commercial products, a topic that will require further study.

ACKNOWLEDGMENTS

This work was supported in part by a JSPS KAKENHI grant (18H03043). We acknowledge the assistance of the Research Equipment Sharing Center at Nagoya City University. We thank Mr. Hiroshi Takase, affiliated with the Center, for his assistance with the SEM.

Conflict of interest

The authors declare that there is no conflict of interest.

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